Thyrotropin-releasing hormone (TRH) analogues offer potential treatment of various maladies of the central nervous system (associated mainly with cholinergic hypofunction due to motorneuron diseases, Alzheimer's disease, electroconvulsive shock therapy, etc.). The main objective of the project is to develop and evaluate chemical delivery systems for targeting centrally active analogues into the central nervous system (CNS) by a chemical-enzymatic approach. Due to covalently attached lipophilic functional groups (a 1,4-dihydrotrigonellyl and a lipophilic moiety) a "packaged" peptide crosses the BBB by passive transport and, once in the CNS, converts to an ionic trigonellyl derivative that is retained at the target site. Then, the biologically active peptide is obtained by sequential metabolism. New brain-targeting systems will be designed and synthesized based on systematically modifying a lead compound ([Leu2]TRH) to improve sequestration of the analog in the brain and/or enhance post-delivery stability of the biologically active peptide. These modifications will allow for a decrease of the systemically administered dose and also for an increase in the residence time of the experimental or therapeutic agent in the CNS. Our hypothesis is that the efficacy of CNS-sequestration can be improved by using alpha-hydroxyglycine to achieve carboxy-terminal amidation via peptidylamidoglycolate lyase (PGL, EC 4.3.2.5) action because of the higher rate of enzymatic bioactivation after esterase cleavage of the protecting ester function. Analogues in which the carboxy-terminal prolinamide is replaced by L-pipecolic acid or the amino-terminal pyroglutaminyl residue is subtituted by an unnatural moiety will also be incorporated into appropriate targeting systems. The design and development will be supported by theoretical calculations. The newly designed analogs will be tested for binding to brain TRH receptors to compare their intrinsic activity with that of the lead compound. In vitro stability and metabolism experiments will address optimization and practical development. Stability studies in brain tissue will address rates, sites and extent of peptide activation and/or cleavage to probe crucial steps in the CNS-sequestration. In vivo distribution and metabolism studies will assess the efficacy of the strategy to transport and sequester the TRH analogs in the brain. We will examine pharmacokinetics of brain-delivery, "lock-in" of the predicted precursors, and the release of the biologically active peptide after parenteral administration of the synthesized targeting systems. Comparative pharmacodynamic evaluation of the effect of brain-delivered analogues will be addressed via in vivo cerebral microdialysis studies in which changes in acetylcholine levels due to treatment will be assayed. Ultimately, pharmacological experiments will be carried out in animals to survey the potential of the approach to treat maladies associated with the loss of cholinergic functions. The antagonism of pentobarbital-induced sleeping will be used as general paradigm to assess the acute effects of brain-targeted compounds. Behavioral observations, dose-dependence and duration of action will also be addressed by appropriate method or study designs.